Fig 2: SARS-CoV-2 E protein priming of monocytes is dose dependent(A) Heatmap of secreted cytokine levels represented as z-scores after human monocyte primary stimulation with mock (n = 4) or E protein stimulation at 1 μg/mL, 10 ng/mL, 1 ng/mL or 10 pg/mL dose (n = 4 at each dose) followed by secondary stimulation with LPS. Hierarchical clustering of analyte rows using 1-Pearson metric.(B) Bar graph showing log 2-fold change of each condition compared to control (mock activated) conditions for individual analytes.∗p ≤ 0.05, Paired Wilcoxon Test. Error bars are standard error of the mean. Analytes ordered by descending Log2FC of highest-dose-treated samples.

Fig 3: CEH enhances intestinal mucosal immunity. ELISA detected the peripheral blood immune factors, TNF-α (A), TGF-β (B), IL-2 (C), and IL-10 (D). (E) Western blot analysis of the TLR4 signaling pathway-associated proteins in small intestine tissue: NF-κB, TLR4, Myd88, JNK, and p-JNK. β-Actin was used as the protein loading control. MPO analysis was used to characterize neutrophil aggregation in the small intestine (F) and spleen (G). (H) HandE staining of paraffin sections for small intestine tissue. (I) Immunohistochemical staining of IgA. Error bars represent mean ± SD. N, negative control; M, model control; CDDP, CDDP chemotherapy; CEH, cepharanthine hydrochloride chemotherapy; Comb, combined chemotherapy strategy; ns, no significant difference. ***P < 0.001.

Fig 4: Comparisons of noise-induced monocyte infiltration and transformation between wild-type and Tlr4 knockout mice. Monocytes and macrophages were identified by immunolabeling CD45, a marker of bone marrow–derived immune cells. (a) The average number of CD45-positive cells in wild-type and the Tlr4−/− mice before and 4 days after acoustic injury. Before noise exposure, tissue macrophages are present in both wild-type and Tlr4−/− mice. There is no significant difference in the number of macrophages between these two strains of mice (two-way ANOVA, P>0.05). Four days after acoustic injury, the numbers of cells are significantly increased in both strains (two-way ANOVA, Tukey pairwise comparison, P<0.05). However, there is no significant difference between the two types of mice (P>0.05). (b and c) Representative images showing the morphology of monocytes and macrophages in wild-type cochleae examined before (b) and 4 days after acoustic injury (c). Double arrows point to the round CD45-positive cells, which exhibit monocyte morphology. Arrows point to the CD45-positive cells showing an elongated cell body, a phenotype typical of monocyte transformation to macrophage. (d and e) Typical images showing the morphology of monocytes and macrophages in Tlr4-null cochleae examined before (d) and 4 days after acoustic injury (e). Double arrows point to the cells with monocyte morphology, and arrows indicate the cells with a transforming monocyte phenotype. Bar=20 μm

Fig 5: Comparison of transcriptional expression of MHC II-encoding genes between wild-type and Tlr4−/− mice. (a) Comparison of the expression levels of H2A-a, H2-Eb1 and H2-M3 before and 4 days after acoustic injury in wild-type mice. There is a significant increase in the expression levels of H2A-a and H2-Eb1 after acoustic injury (Student's t test; P=0.015 for H2-Aa; P=0.017 for H2-Eb1). (b) Comparison of the expression levels of the three genes before and 4 days after acoustic injury in Tlr4−/− mice. There is no significant increase in the expression levels of all examined genes after acoustic injury (Student's t test, P>0.05)